The ICT group demonstrated 10.8% and 29.6% improvement in trunk flexion and body-lift (p = 0.029–0.037 with no improvement in body lock-off and rotation (p = 0.101–0.343). The DCT group demonstrated 5.0–14.9% improvement in the core strength tests (p = 0.012–0.043), a non-significant 33.8% improvement in body-lift (p = 0.100) and no improvement in body lock-off (p = 0.943). In conclusion, none of the training groups demonstrated greater improvement than the other and both dynamic and isometric core training improved climbing-specific test performance. Dynamic training was slightly more favorable although not significantly superior to isometric core training in improving core strength.This is the first study to compare the effects of isometric vs. dynamic core training and characterize core-training adaptations using climbing-specific performance and core strength tests in elite climbers. The aim of the study was to compare the effects of attending a progressive core-training program on climbing performance. 19 advanced and elite climbers  were randomized into a dynamic (DCT) or isometric (ICT) core training group and trained twice weekly for ten weeks. The climbers were tested using two climbing-specific core tests (body lock-off and body-lift) and four non-specific core strength tests—one dynamic (superman) and three isometric (trunk flexion and trunk rotation left and right). Between group comparisons showed no differences between the groups at post-test ( In indoor climbing competition, a route consists of 20–50 holds and takes 2–7 minutes to complete at intensities ranging from 71–91% of maximum heart rate . In rece2 = 0.66–0.76) [Several cross-sectional studies have reported strength and/or endurance in the fingers, body composition and relative upper-body strength as the most important physical factors predicting climbing performance , 5. Seve66–0.76) .The difficulty level of a route is manipulated by several factors such as grip type (size and shape), distance and position between grips and gradient of the climbing wall. As a result, climbing includes dynamic and often explosive movements with short static positions before the next transition. In elite climbing routes (24–27 IRCRA), the climbers often only have two contact points in the wall . To be successful in maintaining positions, reaching climbing holds, clipping the rope to carabiners and moving efficiently, core stability and strength have often been proposed as key factors in elite climbing performance, and although this has never been systematically examined, Muehlbauer and colleagues demonstrated 19–22% increase in core strength (flexion and extension) among sedentary adults after training climbing twice peer week for eight weeks . A stronStudies have examined core training and performance in running , 11, swiIn the majority of sports, both dynamic and isometric core movements are important . PreviouThe study was a within- and between- groups comparison design. Nineteen climbers were randomized to participate in a ten weeks progressive core-training program consisting of either isometric or dynamic core exercises. All participants were tested pre- and post- intervention using one dynamic and one isometric climbing-specific core test, one dynamic core strength test and three isometric core strength tests. During the intervention, the climbers continued their normal climbing routines.p = 0.552–0.847). For details, see Nineteen climbers  volunteered to participate in the study. The climbers  had a mean climbing experience of 7.3 ± 5.6 years. After the pre-test, participants were first stratified (by sex) and than ranked by climbing skills (red-point on-sight climbing performance last year) into pairs . The climbers in each pair were then randomized by drawing into either an isometric core-training program (ICT) or a dynamic core-training program (DCT). Nineteen climbers completed pre- and post- testing . There were no differences between groups in climbing experience, climbing skill or the mean anthropometric measurements at pre-test  according to the IRCRA scale .th Declaration of Helsinki. Participants were informed  about all testing and training procedures and gave written informed consent to participate prior to entering the study. Participants’ gave their written informed consent (in accordance with PLOS consent guidelines) for their images to be reproduced in this manuscript.The study was approved by the Regional Committees for Medical Health and Research Ethics in Norway (2009/1735/REK Sør-Øst D) and conformed to standards of treatment of human participants in research as outlined in the 5Before the pre-test, a pilot study into the test battery was conducted, in which the distance and height difference (i.e. test angle) between hand grip and foot placement in the two climbing-specific core strength tests (body lock-off and body-lift) were examined. With reduced distance or greater height difference between hand and foot placement, grip endurance (rather than core strength) was found to be the limiting factor. Therefore testing procedures were carefully selected to ensure that core strength and not grip endurance was the factor under examination. Core strength tests in neutral spinal and pelvic alignment were also included to test the effect of dynamic and isometric contractions in this position. The exercise known as ‘Superman’ (dynamic version of the prone bridge) has been used in interventions previously , 25, butIn both the pilot and the experimental study, the climbers refrained from strenuous training targeting the upper-body  48 hours before testing. Pre- and post-tests were conducted at the same time of the day. The test-order was randomized for each climber. Before starting the test, the climbers performed a non-strenuous 10-minute aerobic warm-up , 10 minutes of non-demanding traversing of the climbing wall and five boulders (5–10 moves) with medium grips. During testing, 10 minutes recovery between the body-lift and body lock-off tests was permitted and three minutes recovery between the dynamic (Superman) and the isometric core strength tests, with 1–2 minutes recovery between attempts of this tests. Two attempts of the body lock-out and body lift were performed and three attempts of the Superman and isometric core strength tests. Finally, we tested digital strength and endurance in the finger hang test. During testing, participants were verbally encouraged to maximize their performance.The participant adopted a horizontal position with their preferred foot on a campus rung  20 cm above the ground with the curved edge facing down . The parThe climbers used a shoulder-wide pronated grip to hold a 6 cm fixed beam attached 225 cm above the ground . A foot The participants adopted a push-up position with their hands on pads on a slide board  with feet against a wall and the pelvis in a neutral tilt. The climbers were asked to slide their arms forward as far as they were able to and return to the starting position while maintaining the alignment (height and tilt inclination) of their pelvis see . The disThe tests were performed sagittally (trunk flexion) and with rotation to both sides , with thThis test measures isometric finger strength and/or endurance and has been applied as a climbing-specific test in several studies which offer more detailed description , 4, 6. BThe isometric and dynamic training programs were performed twice per week for ten weeks. Each session lasted 30–40 minutes and contained four exercises. The training programs were similar in intensity (4–10 repetitions maximum), volume (3–4 sets) and rests between sets and exercises (2 minutes). The exercises were: foot-lift , arm locThe progression of the training programs was achieved by increasing the number of repetitions, sets, steepness of the wall, decreasing the base of support, increasing the level of the arm(s) and increasing the distance to the foot stance or hand grip. The exercises in ICT were identical, but instead of moving dynamically from a starting to a finishing position as the DCT did, the ICT performed an isometric contraction in the finishing position and maintained the position for three to five seconds, with three seconds recovery between each repetition. If a participant managed to perform the final set with the maximum number of repetitions (DCT) or for the maximum time (ICT), they progressed to a higher level at their next training session. The details of the different intensity levels are presented in p≤0.05; all results are presented as mean±standard derivation. To quantify the strength of the findings, Cohen`s d effect size (ES) was used. An ES of 0.2 was considered a small effect, 0.5 a medium effect and 0.8 a large effect [To assess differences in performance, we used a two-way (groups x time) within-between analysis of variance (ANOVA) with repeated measures. When a significant interaction was detected by ANOVA, paired t-tests with Bonferroni post-hoc correction were applied to locate where the differences lay. Tests were analysed using the SPSS  statistical software package and were analysed per protocol. The significance level was set at e effect .p = 0.931) or post-test (p = 0.592). The ICT group demonstrated a non-significant 10.6% improvement duration of the test  while the DCT had a -0.5% reduction (p = 0.943) between pre-and post-testing (p = 0.817).There was no difference between the groups at pre- (-testing . None ofp = 0.372) or post-test (p = 0.328) in numbers of successful lifts. The ICT improved by 29.6%  and DCT by 33.8% , however the increases were not significantly greater than the other group  or posttest (p = 0.824). A 2.4% and 5.0% improvement was observed for the ICT group  and DCT group  (p = 0.457).There was no difference between the groups at pre- ( = 0.68) . None ofp = 0.388–0.636), rotation right (p = 0.555–0.782) or rotation left , but neither of the groups had greater improvement than the other (p = 0.694). For core rotation right in DCT, there was an 8.2% improvement  compared with 5.3%  in ICT whereas in rotation left in DCT the improvement was 14.9%  compared with 7.4% p = 0.343, ES = 0.31) in the ICT group.No differences between the groups were observed at pre- or post-test in abdominal flexion . No difference in test score after the intervention resulted between groups (p = 0.897).Test performance improved in both groups by 4.5% ± 16.8 in DCT and 4.1% ± 20.0 in ICT; however the improvement was not significant  and 25 ± 2 (DCT). In addition, 45 ± 21 (75.1 ± 39.3 hours) and 34 ± 11 (54.4 ± 25.1 hours) climbing sessions were logged  during the intervention.The DCT group demonstrated improvement in three isometric core tests  and in the dynamic core-strength, however, there was no improvement in two climbing-specific core tests. In comparison, the ICT group improved in isometric trunk flexion strength testing and a climbing-specific test (body-lift), involving dynamic trunk and hip activation, with no improvement in the trunk rotation or body lock-off tests. These results are not in accordance with proposals of training-specific adaptations, as the dynamically trained group in this study performed superiorly on isometric strength testing after the intervention, with some evidence of isometrically trained participants performing better on a task with dynamic trunk and hip actions.We hypothesized there would be improvements for both groups in the climbing-specific tests: dynamic training using the exercises chosen (foot-lift and arm lock-off) requires unilateral hip flexion, either whilst the upper body supports the trunk (foot-lift) or during cross-lateral stabilization , as the unsupported upper limb and foot reach for a hold (arm lock-off). Both exercises demand isometric trunk stiffness to provide a base of support for the limb actions, with dynamic motion produced either at the shoulder or pelvic girdle. At the volume prescribed (see Tables In the other climbing specific test (body-lock), neither group demonstrated significant improvement, which is perhaps less surprising as this test is performed horizontally, a position which occurs rarely in climbing . The impTo our knowledge, no previous studies have compared the effect of isolated vs dynamic core training. Some, but not all previous studies have demonstrated improved performance after improved core strength and/or stability , 22, 33.Core strength was measured in isolation with a dynamic test (Superman) and three isometric tests . Neither group demonstrated greater improvement than the other. However, the DCT group performed better in all tests after the intervention, while the ICT group only improved in Superman and trunk flexion. It is possible that differences in exercise performance intensity (dynamic versus isometric) could have influenced results, however, total exercise duration was matched according to previous approaches . FurtherIn contrast to the principle of training-specificity  and taskThe study has some limitations: only eighteen climbers completed the study (nine participants in each group), which increases the risk of a type II error due to low statistical power. Also as core-training was not performed in isolation, but included within participants’ overall fitness routine (i.e. climbing and bouldering), the impact of non-specific trunk loading as a result of other training activities may have had an effect on results . HoweverThis study is the first of it is kind to compare the effects of dynamic and isometric core training in elite climbers and to examine whether these modes of core-training improve climbing performance. Although neither training approach demonstrated significantly greater improvement overall, the DCT improved in both sagittal and rotational core strength tests, whereas the ICT only improved in the sagittal test. Both modes of training appear equally effective in improving sagittal MVC. With regard to climbing-specific tests, only the ICT group demonstrated an improvement after the intervention, however this finding should be interpreted with caution, as a positive association between improvement in core strength and climbing performance may be harder to detect in highly trained climbers with high core strength at baseline. In summary, based on findings from this and previous comparable studies , 11, 13,S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Figp<0.05.* Significant difference between pre-and post results, (TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Switch-associated protein 70 (SWAP-70) is a guanine nucleotide exchange factor that is involved in cytoskeletal rearrangement and regulation of migration and invasion of malignant tumors. However, the mechanism by which SWAP-70 regulates the migration and invasion of glioblastoma (GB) cells has not been fully elucidated.This study used an online database to analyze the relationship between SWAP-70 expression and prognosis in GB patients. The in vitro wound healing assay and transwell invasion assay were used to determine the role of SWAP-70 in GB cell migration and invasion as well as the underlying mechanism.We found that patients with high SWAP-70 expression in the GB had a poor prognosis. Downregulation of SWAP-70 inhibited GB cell migration and invasion, whereas SWAP-70 overexpression had an opposite effect. Interestingly, SWAP-70 expression was positively correlated with the expression of the standard form of CD44 (CD44s) in GB tissues. Downregulation of SWAP-70 also reduced CD44s protein expression, whereas SWAP-70 overexpression enhanced CD44s protein expression. However, downregulation of SWAP-70 expression did not affect the mRNA expression of CD44s. Reversal experiments showed that overexpressing CD44s in cell lines with downregulated SWAP-70 partially abolished the inhibitory effects of downregulated SWAP-70 on GB cell migration and invasion.These results suggest that SWAP-70 may promote GB cell migration and invasion by regulating the expression of CD44s. SWAP-70 may serve as a new biomarker and a potential therapeutic target for GB. Glioblastoma (GB) is the most common and malignant type of brain tumor in adults. GB is a highly invasive tumor and its mortality is among the highest of all malignancies in humans , 2. AlthCellular invasion is a complex cellular process that includes cell-to-cell and cell-to-extracellular matrix (ECM) interactions, as well as cellular migration and degradation of ECM enzymes –7. The mSWAP-70 is a phosphatidylinositol triphosphate (PIP3)-binding protein, belonging to the family of guanine-nucleotide exchange factors (GEFs) . SWAP-70The present study investigated the roles and underlying mechanisms of SWAP-70 in GB cellular migration and invasion. We found that high SWAP-70 expression in high-grade glioma tissues was associated with a poor prognosis of glioma patients. We also found that SWAP-70 regulated the expression of the standard form of CD44 (CD44s) to promote GB cellular migration and invasion. Collectively, our findings provide an experimental basis for the applicability of SWAP-70 as a potential therapeutic target for GB.A total of 62 patient tissue samples including 15 cases of non-tumorous brain tissues and 47 cases of gliomas tissues  were used in this study. All the specimens were obtained from the Affiliated Hospital of Xuzhou Medical University . Glioma patients were histologically diagnosed according to World Health Organization (WHO) criteria. None of the patients received any therapies before sample collection, such as radiation, immunotherapy or chemotherapy. Written informed consent was obtained from all of the patients, and this study was approved by the Research Ethics Committee of the Affiliated Hospital of Xuzhou Medical University. All the tissue samples were frozen in liquid nitrogen.Total protein extracts from non-tumorous brain tissues and glioma tissues (WHO II-IV) were subjected to Western blot analysis as described previously . The exp2. Antibodies against SWAP-70 (ab89532) and CD44s (ab51037) were obtained from Abcam. Antibodies specific for β-actin (A1978) and Flag (F7425) were purchased from Sigma.Cell lines 293T, U251 and U87 were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences for this study. These cell lines were cultured in DMEM supplemented with 10% FBS at 37 °C in a humidified incubator with 5% COFor SWAP-70 silencing, two short hairpin RNA (shRNA) duplexes were designed to target human SWAP-70 gene. The shRNA oligomers were annealed and then subcloned into the pLB plasmid by the HpaI and XhoI cloning site. For overexpression of SWAP-70, the cDNA encoding human SWAP-70 gene was inserted into the pWPXLd-puro lentiviral vector using BamHI and MluI sites. For silencing of CD44s, a pair of shRNA sequences were synthesized and incubated in annealing buffer. Annealed shRNA oligomers were inserted into pLVshRNA-EGFP plasmid digested with BamHI and EcoRI. The CD44s gene fragment was obtained by PCR amplification with the unique restriction sites EcoRI and NotI. After amplification, the PCR product was inserted into the lentiviral vector pLV-EGFP-N. The primers for CD44s overexpression contained a Flag tag. The primers used in this study were shown in Table A lentiviral packaging system was used in this study as previously described . BrieflyCell migration was assessed by a scratch wound assay. Briefly, cells were seeded in 6-well plates and grown to confluency. A scratch wound was created using a plastic pipette tip, detached cells were washed away with PBS, and then the remaining cells were incubated in serum-free media. After 24 h and 48 h incubation, five random fields were photographed using an Olympus IX-71 inverted microscope. The number of migrating cells across the wound was counted on these images.Cell invasion assay was performed using a transwell system. Diluted Matrigel with cold distilled water was applied to 8 μm pore size polycarbonate membrane filters. Cells in serum-free medium were plated added to the upper chamber at a density of 10,000 cells/well. In the lower chamber, the DMEM media containing 10% FBS was added. Cells were incubated for 36 h and then the non-invading cells were removed with cotton swabs. Cells that invaded to the bottom of the membrane were fixed in 4% methanol for 20 min and stained with a 0.3% crystal violet solution for 30 min. The number of invading cells was analyzed by a microscope.Total RNA was extracted from control and SWAP-70-downregulated cells using Trizol (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was performed by Transcriptor First Strand cDNA Synthesis Kit (Roche). qPCR was performed as our previous study protocol . The folP < 0.05 were considered to be statistically significant.Each experiment was performed at least three times. Results are expressed as the mean ± SEM. The statistical analysis was performed by using GraphPad Prism 6.0 software. Differences between control and experimental groups were assessed by Student’s t-test or One-way ANOVA. To study the role of SWAP-70 in the pathogenesis of glioma, Western blot analysis was used to measure the SWAP-70 protein expression in glioma tissues and non-tumor brain tissues. As shown in Fig. SWAP-70 gene sequence-specific short hairpin RNA (shRNA). The effects of SWAP-70 downregulation on GB cellular migration and invasion were tested using an in vitro scratch assay and transwell invasion assay. First, GB U87 and U251 cell lines with stably downregulated SWAP-70 expression were constructed. Western blotting was used to detect the downregulating efficiency of SWAP-70. As shown in Fig. To investigate the role of SWAP-70 in GB cellular migration and invasion, SWAP-70 expression was downregulated in GB cells using Results of the in vitro scratch assay showed that the wound healing rate of the control group was faster than that of the SWAP-70 downregulation group at 24 and 48 h after scratching. Statistical analysis showed that after SWAP-70 downregulation, the migration rates of U251 cells decreased by approximately 40% and 57% at 24 and 48 h, respectively, after cell scratching. The migratory rates of U87 cells decreased by approximately 35% and 52% at 24 and 48 h, respectively, after cell scratching  [CD44s but regulated the expression of CD44s protein. The dissociation of CD44 is regulated by protein kinase C, calcium ion flux, and small G-proteins of the Ras homologous (Rho) family. The Rho family molecules recruit metalloproteinases to surround CD44, promote CD44 dissociation, and alter the CD44 distribution on the plasma membrane [CD44 is a transmembrane glycoprotein that is expressed in the plasma membrane. The human ed exons . CD44 mo (CD44v) . The str (CD44v) . CD44 is (CD44v) , 37. Abn (CD44v) , 38, 39. (CD44v) . CD44s p (CD44v) , 26. In  (CD44v) . The intmembrane . SWAP-70Taken together, the results of this study demonstrate that high SWAP-70 expression is a biomarker for poor prognosis of GB. SWAP-70 plays an important role in GB cellular migration and invasion. Importantly, SWAP-70 promotes GB cellular migration and invasion by regulating CD44s expression. Thus, SWAP-70 may serve as a novel biomarker and potential target for the treatment of human GB.


Psychosocial screening in brain tumor patients is of high importance. We applied The Basic Documentation for Psycho-Oncology Short Form (PO-Bado SF) in primary brain tumor patients and patients with metastasis. The aim was to evaluating consistency between physicians' perception and the results of the patients' self-assessment.140 patients with first diagnosis of a brain tumor were screened during their hospital stay (t1) using Distress Thermometer (DT) and Hornheide Screening Instrument (HSI), health-related quality of life was assessed by EORTC QLQ-C30 + BN20. After 3 (t2) and 6 months (t3), patients were re-evaluated. Attending neuro-oncologists completed the PO-Bado SF at all three time points (cut-off for being in need for support >8).At t1, the mean of the PO-Bado SF total score was 7.71 (SD = 4.08), at t2 8.22 (SD = 5.40) and at t3 7.62 (SD = 5.72).r = 0.6, p < 0.0001) and lower EORTC GHS scores .The proportion of patients reaching a total score >8 was at t1: 43%, at t2: 41% and at t3: 47% (t1–3). Discrimination of PO-Bado SF total score, between patients in (DT ≥6) and those not in distress was more sensitive  than discrimination compared to the HIS . Higher PO-Bado-SF total score correlated with higher DT scores (Physicians' perception according to PO-Bado SF provides a different measure for psychosocial burden in patients with brain tumors, however does not completely reflect patients' wishes. Recen [et al. ). Theref [et al. , 10.However, feasibility and acceptance by both patients and health professionals remains suboptimal . Due to et al. conducted an analysis comparing – inter alia - the Distress Thermometer (DT) values of 1551 cancer patients  and the results of PO-Bado SF results scored by physicians and found a total score of 9 or greater to be optimal [Therefore, the assessment of psychosocial distress by interviewers (ClinRO) may provide an important addition to screening for psychosocial burden in patients who are unwilling or unable to fill in self-report questionnaire. The Basic Documentation for Psycho-Oncology Short Form (PO-Bado SF) is a clinician-administered instrument to guide professionals in a focused and structured psycho-oncological assessment developed from The Basic Documentation for Psycho-Oncology . It has  optimal . HoweverIn our prospective observational study we evaluated the psychosocial burden during the early disease trajectory of brain tumor patients six months after first diagnosis by self-reporting questionnaires but also by the PO-Bado-SF as an expert rating scale.We investigated 1) the comparability between physicians’ perception of the patients’ burden and the results of the patients’ self-assessment, 2) if the PO-Bado SF total score cutoff described in the literature can be applied to brain tumor patients as well and 3) the capability of the PO-Bado SF global burden (GB) as single item to reliably identify need for psychosocial support/distress.n = 76, 54%) as shown in Figure n = 122, 87% and postoperatively n = 128, 92%). Most of the patients suffered from malignant gliomas (37%) and meningiomas (31%). Metastasis from solid tumors occurred in 22%. Most of the tumors were located in the frontal lobe . In the majority of cases a gross total resection (GTR) could be achieved . All patient and tumor characteristics as well as demographic data are displayed in Table n = 23 patients and between t2 and t3 further n = 20 dropped out, n = 10 died until t2 and n = 8 until t3. At t2 n = 9 and at t3 n = 4 were excluded due to incompliance to assessment (did not fill in the questionnaires as scheduled).Data of 140 patients recruited between September 2012 and September 2014 was analyzed with a slight female predominance in both centers . Mean time of PO-Bado SF assessment was 7.5 min (range 5–18.5 min). At t1, the mean of the PO-Bado SF total score was 7.71 , at t2 8.22  and at t3 7.62 .The PO-Bado SF interview was conducted in n = 60 out of 139 (43%), at t2 n = 49 out of 117 (41%) and at t3 n = 45 out of 96 (47%).The proportion of patients reaching a total score ≥8 was at t1 n = 20 (14%) patients were estimated as being in need of support by the clinicians consistently at all three time points  and n = 26 with complete FU were assessed by a total score ≥ 8 at t1 and t3 (Table Only t3 Table .The item means range between the highest 1.80 (SD = 1.27) for “fatigue/tiredness” at t2 and the lowest 0.83 (SD = 1.2) for “other problems, e.g. social or family problems” also at t1.Results are displayed in more detail in Table Significant differences between tumor entities were only seen for PO-Bado SF GB scores postoperatively and at t2 for PO-Bado SF total score by univariate explorative analyses. Complete results are shown in the Supplementary .n = 51 (36%) patients as being in need of support. After 3 and 6 months n = 29 (25%), resp. n = 22 (23%), scored ≥6 on the DT. With the HSI, comparable percentages of patients in need of psychosocial help were identified by the screening .At t1, DT identified DT-Scores improved over the course of the investigation with significant differences between assessments. The need for treatment based on the HSI was comparable between assessments.p < 0.001), and regarding the HSI in 46–61%  of the cases. Further, n = 34 (24%) patients requested psychological support after diagnosis was confirmed (t1) of which 19 (80%) were identified by physicians via PO-Bado-SF as being in need for support at the same time. After 3 resp. 6 months, n = 12 resp. n = 11 patients (10% resp. 11%) immediately asked for psychological help, by PO-Bado-SF n = 8 (67%) resp. n = 6 (54%) of these patients were detected as in need for support. Similarly, the DT results and HSI results were only partially in agreement. Further detailed information on DT and HSI results as well as agreement between results of external assessment and self-reporting questionnaires are shown in Table PO-Bado SF showed only fair agreement with the screening results of DT and HSI regarding patients’ need for psychosocial intervention : RegardiUsing the PO-Bado SF total score, discrimination between patients in and those not in distress (DT ≥ 6) showed a greater sensitivity  than discrimination between patients in need of treatment according to the HSI, which however turned out to provide a better specificity . More results of the receiver operator characteristic analyses utilizing PO-Bado-SF total score are displayed in Table The PO-Bado-SF GB showed a better discrimination between patients in distress and those not in distress (DT ≥6) at a cut-off of 4.5  than in patients in need or not in need of treatment according to their HSI results . Sensitivity for correct discrimination was moderate with the highest sensitivity and specificity at 6 months for both variables. Further information on the results of the ROC-analysis can be seen in Table Median patient reported GHS ranged between 50 (t1) and 75 at t3 (range 0–100). Detailed EORTC results are displayed in A large correlation was found between PO-Bado-SF total score and GB, as well as between both general PO-Bado Scores and DT and EORTC QLQ-C30 GHS, respectively Table . The detIn our study we applied the PO-Bado SF in brain tumor patients during the early disease trajectory after first diagnosis. We evaluated the interviewer-based expert rating for distress screening in cancer patients longitudinally in a sample of 140 patients. Our analyses revealed that a relevant proportion  of brain tumor patients reached a total score > 8  during the study. In some cases due to the mentioned questions the patient-doctor consultation was prolonged. This has to be taken into account regarding feasibility in clinical routine. Further, the application was in a patient subgroup fitting into the study inclusion criteria what leads to a certain selection bias. However, also when implementing PRO measures in order to screen for distress the indicated problems or items mentioned by the patients have to be discussed what can prolong patient-doctor consultation. Finally, the implementation of ClinRO has to be proven in clinical practice, although we feel that in brain tumor patients they may be useful.In line with the results of Marten-Mittags, the item with the lowest mean was “other problems” and the item with the highest mean was “anxiety/worries/tension” postoperatively. However, after three and six months “fatigue/tiredness” became of greatest importance. This emphasizes that fatigue plays a major role for brain tumor patients, which is reported also by patients themselves in up to 90% –23.The mean total score of PO-Bado SF in our study was comparable (mean of t1-t3: 7.89) to others –19. The Interestingly, the PO-Bado SF total score showed the highest peak at t2 with high item means in “fatigue/tiredness”, “function” and “anxiety/worries”. As also reported by others, patients with brain tumors, who undergo radiation after surgery, show high symptom burden right after the treatment, which improves over time , 27. HowAdditionally to the different screening measures, we investigated the relation between PO-Bado SF and the EORTC QLQ-C30/BN20, because we feel that distress and perceived health related QoL are closely linked, what has been shown in previous studies , 29, 30.Further, GHS, emotional functioning and future uncertainty showed large correlations with PO-Bado total score and GB, whereas other functioning scores  demonstrated only moderate correlations with both PO-Bado, what may be explained by the heterogeneous patient cohort . InteresPsychosocial screening instruments–either self-reporting (PRO measures) or expert-rating scales (ClinRO) – are developed in order to divide patients into groups either in or not in need of psychosocial support, thus cutoff scores are required. With regard to PO-Bado SF total score, cutoff scores >8 and >9 were reported: Marten-Mittag found with their cutoff >9, that 36% of their heterogeneous patient sample suffering from clinically relevant distress , 18. OurUsually, psychosocial screening is performed by applying short self-reporting questionnaires or screening instruments. However, the topics patients most require help with may not be always the topics that physicians or other health care professional feel necessary or even able to address . This unFinally, taken into account the critical aspects of external assessment, the PO-Bado SF turned out to be implementable in the consultation in our study. Even if the evaluation of the full length interview during daily routine is not always feasible, using the global burden (GB) as indicator if psychosocial distress is present seems to be reliable, as we have found a large correlation between the SF total score and physician assessed global burden (GB). Optimally the assessment comprises both: self-reporting instruments and external assessment. Finally, a low-threshold offer of psycho-oncological interventions should be available and the provision of psychosocial support should not be based solely on self-reporting or external assessment in brain tumor patients.n = 3) with difference in their clinical experience conducted and rated the PO-Bado SF, which may have influenced the ratings, but again the results are therefore more applicable to daily routine with physicians at different levels of training treating and screening patients. As the interviewers of PO-Bado were also the treating physicians, they were aware of the patients’ history, prognosis and further therapies, which might also have biased the evaluation. Moreover, we observed a high drop out of the study, partially due to death indicating the severity of the disorder. However, in observational studies assessing patient reported outcomes, drop-outs are frequently [Off note, our study has some limitations: due to the inclusion criteria, we have to assume a certain selection bias as only patients were included being able to fill in questionnaires. Second, we included patients with first diagnosis of any intracranial tumor, leading to a heterogeneous patient sample with primary and secondary, malignant and benign tumors. At the same time, it is one of the strength of the study, as the complete spectrum of patients in neuro-oncological units is reflected by the results. Third, several interviewers  were recruited after surgery and histopathological confirmation of the tumor entity as shown in Figure Informed consent was obtained after surgery by . Each patient was screened three times for psychosocial burden, general health perception, health-related quality of life (HRQoL) and distress using the NCCN Distress Thermometer  (DT), thScreenings were performed postoperatively during the inpatient period at day 5–7 after surgery once the patient had been informed about the tumor’s entity, as well as at three and six months postoperatively during the routine outpatient visits. In case of benign neoplasms the third assessment was performed over the phone and questionnaires to be filled in by the patients were sent by mail free of charge for each patient. When sending the questionnaires by mail, patients had to return them within 2 weeks. If not they were reminded by the study members by a phone call. When questionnaires were not returned within 4 weeks, patients were excluded from the study.Additionally, demographic and tumor specific data  and pre- as well as postoperative Karnofsky Performance Scale (KPS) scores were collected. A flow chart of the study design is provided in Figure http://www.po-bado.med.tum.de/). The clinical cutoff criteria indicating need for psychooncological intervention proposed by Herschbach et al. are the following: a) at least one of the six PO-Bado SF items is scored 4, b) two items are scored 3. Alternatively, a PO-Bado SF total score of ≥8 is defined as the second cutoff criterion [The Basic Documentation for Psycho- Oncology Short Form (PO-Bado SF) is an external assessment tool for oncologist developed from the PO-Bado standard version , 17 in oriterion , 17. In The National Comprehensive Cancer Network (NCCN) Distress Thermometer (DT) measures distress of cancer patients in terms of a single item presented as an 11-point visual analogue scale with scores from 0 (not distressed) to 10 (extremely distressed) along with further 40 items concerning financial, physical, emotional and spiritual concerns , 41–43. The European Organization for Research and Treatment of Cancer Quality of life core Questionnaire (EORTC QLQ-C30) is a frequently used patient reported outcome measure including 30 items to assess health-related quality of life in oncological patients in terms of functioning and symptom burden . The iteet al., is provided in order to screen patients in need of psychosocial support [et al. and our group recently in brain tumor patients [The Hornheide Screening Instrument (HSI) was initially applied to evaluate psychosocial burden of skin tumor patients and developed from the Hornheide questionnaire , 45. The support . Althougpatients , 47.Primary outcome was correlation between PO-Bado SF total score as well as PO-Bado SF GB and self-reporting instruments  and to evaluate the PO-Bado SF total score and GB cutoff for brain tumor patients based on DT and HSI with respective sensitivity and specificity.Secondary outcomes were results of the PO-Bado-SF assessment at t1-t3 in patients with intracranial tumors and agreement between external assessment und self-reporting questionnaires concerning need for support/clinically relevant distress. Agreement in PO-Bado SF with self-reporting questionnaires was defined as follows:PO-Bado SF with DT: agreement +/+ = Patients with PO-Bado SF total score ≥ 8 and DT ≥ 6, agreement −/− = patients with PO-Bado SF total score <8 and DT<6.PO-Bado SF with HSI: Agreement +/+ = PO-Bado SF total score ≥8 and patient in need of psycho-oncological support according to the discriminant analysis tool as well as agreement −/− = PO-Bado SF total score <8 and patient in not need of psycho-oncological support according to the discriminant analysis tool.Cohen’s Kappa statistics were performed to assess agreement. κ = 0.01–0.2 was defined as slight, κ = 0.21–0.4 as fair, κ = 0.41–0.60 as moderate, κ = 0.61–0.80 as substantial and κ = 0.81–0.99 as almost perfect agreement .U-Test or Pearson-Chi-Square test as appropriate based on the measurement level.Demographic and tumor-related data as well as KPS were analyzed descriptively. Differences in distribution between study centers were estimated using Mann-Whitney-Results from the questionnaires were tested for normal distribution using the Kolmogorov-Smirnov-Test. Pearson or Spearman-Rho-Correlations between DT-scores/EORTC functioning and symptom scores and PO-Bado scores were used as appropriate according to the data’s distribution. Correlations >0.5 were described as large, >0.3 as moderate and >0.1 as small . ANOVA wSensitivities and specificities for PO-Bado GB and total score cut-off values based on results of the HSI and DT scores were determined using ROC-Curves. Cut-offs were selected based on the optimal balance between sensitivity and specificity .et al. cumulated their data of patients at different time points [For the ROC- and correlations analyses all assessment were assumed to be independent samples and therefore cumulated, because the current state of psychosocial burden was assessed at each assessment using different screening tools evaluating their agreement and no specific psycho-oncological treatment was initiated in between possibly influencing the following screening. Similarly, Martin-Mittag e points .P < 0.05 was considered statistically significant. All analyses were performed using SPSS, version 18.0 .This study was performed in accordance with the Helsinki Declaration after approval by the local ethics committee (No. 837.220.12 (8321-f)). The responsible clinical investigators  informed eligible patients verbally and handed out written information about the study. Participants provided their written informed consent and were assigned a patient identifier to ensure data confidentiality.The physicians’ perception by the PO-Bado-SF provides a different aspect of psychosocial burden patients with intracranial tumors and cutoffs for brain tumor patients could be elaborated in the study. However, the physicians’ view does not completely reflect the patients’ wishes. Therefore, patient reported outcome measures are indispensable, but should be accompanied by the assessment of the physicians.


Acinetobacter baumannii is an important pathogen in health care and is responsible for severe nosocomial and community-acquired pneumonia. To design novel therapeutic agents, a mouse model for A. baumannii pneumonia is essential. A. baumannii using clinical and 19606R standard strains for developing a quantitative real-time PCR (qRT-PCR) for rapid identification of A. baumannii infection from lung tissues of BALB/c mice. We described a mouse model of 8, 1 × 108, and 1.5 × 108 cfu/ml) were used. Lung tissues were cultured and compared with ompA gene. Clinical isolates had better positive results at day three with the highest dose than 19606 strain either in culture (4 versus 3) or in qRT-PCR (5 versus 4). However, qRT-PCR detection was 100%, the specificity was 70%, and the positive predictive value was 27%. To infect the mice, three doses of bacteria (0.5 × 10A. baumannii in the BALB/c mice model has a higher sensitivity than the culture method.The qRT-PCR detection of  Acinetobacter baumannii is regarded as one of the most important opportunistic pathogens of increasing clinical importance over the course of the last three decades. A. baumannii infection has recently emerged as a major cause of health care-associated  infections worldwide. Pneumonia is the most frequent clinical presentation of A. baumannii infections[3]. This bacterial agent is identified as the fifth most common pathogen in Intensive care units (ICUs) in developed and developing countries. The high rate of antibiotic resistance observed for A. baumannii isolates is attributed to its ability to employ a number of virulence factors such as OmpA. OmpA is highly conserved among bacterial species, and in A. baumannii, it has been associated with a variety of interesting biological properties in in vitro model systems. OmpA has also been associated with antimicrobial resistance in related pathogens[10]. The frequent existence of multidrug-resistant, extensive drug-resistant, and pandrug-resistant A. baumannii isolates revived the use of colistin, as an old polymyxin antibiotic[11-14]. Recently, colistin-resistant A. baumannii isolates are augmenting, which represents extremely alarming phenomenon[15]. Therefore, there is an urgent need for the development of novel therapeutic approaches.A. baumannii pneumonia have been developed and used widely[3]. In these models, several inbred strains of mice such as C57BL/6 and BALB/c as well as A/J strains have been used. Although these strains are likely to demonstrate a more homogeneous response to infection, they may not exactly mirror the variable responses that may take place in humans[16-21]. To recognize the infected mice and verify the production of infectious models, researchers apply diagnostic methods. Current diagnostic techniques rely on culture-based approaches, which are time-consuming and dependent on individual interpretation. In addition, the sensitivity of culture method is not high enough[22]. Nowadays, the real-time PCR is being used for sensitive detection of different viral[23] and bacterial infections[24], which improves the early detection of these diseases. Animal models are essential for the progress towards developing new therapeutics and vaccines and play fundamental roles in the evaluations of efficacy and safety of the new products before entering clinical trials. During the past years, the mice models of A. baumannii in infected lung tissues of BALB/c mice using a clinical isolate and the strain 19606 of A. baumannii.The aim of this study was to optimize a quantitative real-time PCR (qRT-PCR) method for rapid identification of Bacterial strains and culture conditionsA. baumannii isolated from burn-wound infection was used in this study[25]. This strain is resistant to amikacin, cefepime, ceftazidime, ciprofloxacin, colistin, gentamicin, imipenem, levofloxacin, minocycline, tetracycline, tobramycin, and trimethoprim-sulfamethoxazole, but it is susceptible to ampicillin-sulbactam /mL, as verified by both spectrophotometry (OD600 0.01–0.02 nm) and colony counting[27].A CR-XDR ulbactam . The phee strain . Fresh bA. baumannii isolate confirmationA. baumannii using API 20NE system  and was later confirmed by the detection of blaOXA-51- PCR, to differentiate between Acinetobacter calcoaceticus and Acinetobacter genomic species. The clinical isolate was stored at -20 °C in CRYOBANK™  until further use.The isolate was identified as MicePasteur Institute of Iran . Animals had free access to food and water except during experimental procedures. All studies were performed in accordance with the institutional ethical guidelines for the care and use of laboratory animals, and the protocol was approved by Tehran University of Medical Sciences, Tehran, Iran[30]. Eight- to 12-week-old, specific-pathogen free, male or female BALB/c mice, with 25–35 g in weight, were purchased from the Intranasal A. baumannii inoculationA. baumannii isolate as previously mentioned by van Faassen et al.[31]. Mice were anesthetized by intraperitoneal (i.p.) injection of 12.5 mg/kg (5 µl) xylazine and 80 mg/kg (25 µl) ketamine and then inoculated intranasally with appropriate numbers of various A. baumannii isolates in 50 μl of saline. For evaluation of infection using real-time PCR, 180 mice were assigned to four groups (5 mice in each group) using three doses  of bacteria in 50 µl for three days.For intranasal inoculation, fresh inocula were prepared for each experiment from frozen stocks of the Conventional bacterial culture of pneumonic mice lungFive mice of each group were sacrificed 24 hours after days 1, 2, and 3 post inoculation. The lung (about 0.36 g) was removed and segmented and then homogenized under the sterile condition. Next, 1 ml saline was added to the homogenized tissue, and 100 μl was cultured on Muller-Hinton agar (MHA) and then incubated at 37 °C for one day. One part of tissue was removed for qRT-PCR, and the rest was used for the conventional culture. Evaluation of the A. baumannii OmpA gene expression by qRT-PCR DNA extraction from lung tissueA commercial kit  was used in accordance with manufacturer’s recommendations to extract DNA. About 4 g of each tissue was dissected using a sterile scalpel blade and placed in a stomacher bag containing 20 ml of Earle's balanced salt solution. The sample was homogenized with a blender at high speed for 60 s. The resultant supernatant was stored at -20 °C until it was used for DNA extraction. To extract DNA, 100 μl of liquid homogenate was mixed with 180 μl of ATL buffer (Tissue Lysis Buffer), supplied with the kit, and then the kit’s spin-column protocol for “purification of total DNA from animal tissues” was used.Primerset al.[32] are as follows: OmpA-F (TCTTGGTGGTCACTTGAAGC) and OmpA-R (ACTCTTGTGGTTGTGGAGCA) as well as 16sr RNA-F (ACTCCTACGGGAGGCAGCAGT) and 16sr RNA-R (TATTACCGCGGCTGCTGGC). Sequences were checked for specificity using NCBI primer BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). All primers were optimized using temperature gradients, and the PCR products were visualized on 1% agarose gel electrophoresis. The equivalent Tm value for OmpA gene (Tm: 79.4 ± 0.4 °C) was also detected when the positive control strain  was tested with qRT-PCR. Primers used for qRT-PCR, previously published by McConnell qRT-PCR Qiagen Rotor-Gene SYBR Green Kit. A standard curve (using at least 5 standards) as well as positive, negative, and no template controls were included in every run. For SYBR green-based reactions, the conditions were as follows: 10 min at 95 °C, followed by 40 cycles of 5 s at 95 °C and 10 s at 60 °C. At the end of each reaction, a melting curve (from 50 to 99 °C) was performed. In order to rule out the false-negative results due to DNA losses during DNA extraction or the presence of PCR inhibitor in the template, human herpes virus 6 (HHV6) DNA  was sed as an internal control. Mice were scarified at days 1, 2, and 3 post inoculation with three sub-lethal concentrations of the bacteria , and the tissue was evaluated with qRT-PCR OmpA expression.All real-time PCRs were performed on an ABI 7300 qRT-PCR detection system . Real-time qPCR amplification was carried out using the Statistical analysist-test by one-way ANOVA. Differences were considered statistically significant when p ≤ 0.05.Data were presented as means ± standard deviation (SD) for each group. Differences were assessed by student’s Establishment of qRT-PCR assays1 to 106 copies/μL. The limit of detection for the target DNA was 10 copies per 20 μL reaction volume. The assays correlated well for OmpA (r2 = 0.994). Intra- and inter-assay repeatability was tested in triplicate for each dilution within the same run, and each concentration was repeated three different times to assess the reproducibility of the qRT-PCR assays. The coefficient of variation of intra-assay and inter-assay were low and in the ranges of 0.06%–0.48% and 0.08%–4.40%, respectively. Intra- and inter-assay coefficient of variation of less than 4.2% confirmed the high repeatability of the assays. Samples were considered positive if a threshold cycle was reached during the 35 cycles. The linearity and limits of detection of the assays were determined with serial 10-fold dilutions of ATCC 19606R DNA from 10Comparison of conventional bacterial culture results with qRT-PCROmpA sequence of different strains of A. baumannii are similar, the standard curve was used to determine the existence of bacterial genomic DNA in the lung tissue. On third day post infection, the lungs of the infected mice in conventional culture showed the highest positive number when the highest inoculation dose (1.5 × 108 CFU/ml) was used; however, clinical isolate had more positive results than the standard strain  of the bacteria (as OmpA). We also demonstrated a high sensitivity and negative predictive value of direct qRT-PCR detection for identification ofThis report is the first observational study to assess the clinical utility of qRT-PCR on specimens from the respiratory tract of a mouse model for detection of A. baumannii. The results revealed that in both methods , the highest dose (1.5 × 108 CFU/ml) of the bacterial inoculation had the best positive outcomes in all three days post inoculation, especially in relation to the clinical isolate of A. baumannii[22].A. baumannii, as the gold standard, the sensitivity of qRT-PCR detection was 100%, the specificity was 70%, the positive predictive value was 27%, and the negative predictive value was 100% among the samples collected from A. baumannii pneumonic mice. We observed a high frequency of samples that were negative by the conventional bacterial culture but positive by qRT-PCR. One possibility is that some Acinetobacter strains that are detected by qRT-PCR could not able to grow on Acinetobacter medium[24]. The application of qRT-PCR was limited by the low positive predictive value for the following possibilities. First, negative culture results could have been obscured by immune system of mice in some cases. As such, the dead bacterial remnants would fail to grow on culture, but the qRT-PCR detects the presence of genomic DNA in the samples. Second, more sensitive nature of qRT-PCR detection might result in recognizing a small number of A. baumannii, which might not be detected in culture[23]. Third, the PCR might be attributed to a false-positive results, possibly indicating the existence of bacterial DNA that is not associated with viable bacteria[32]. However, the inclusion of a negative PCR control minimized this possibility. Fourth, more sensitive qRT-PCR might detect a small number of A. baumannii, which are not responsible agent for pneumonia.Using positive culture of A. baumannii in pneumonic animal models. However, further studies are required to probe the potential role of this approach. In conclusion, our study indicated that qRT-PCR is a sensitive method for diagnosis of


Curcuma longa is extensively studied. Curcuma raktakanda, a poorly studied species, is most commonly distributed in the Kerala state of India. For the first time, we examined the efficacy of different fractions  of C. raktakanda against glioma, cervical, and breast cancer cell lines. As determined by mitochondrial reductase activity assay, the viability of cancer cells was decreased in a concentration-dependent manner by the three fractions. The half maximal inhibitory concentration (IC-50) values after the treatment of C-6 glioma cells for 48 h was found to be 32.97 µg/mL (acetone extract), 40.63 µg/mL (hexane extract), and 51.65 µg/mL (ethyl acetate extract). Of the three fractions, the acetone fraction was more effective. The long-term colony formation of cancer cells was significantly suppressed by the acetone fraction. Analyses using DAPI  staining, AO/PI (acridine orange/propidium iodide) staining, DNA laddering, and sub-G1 population revealed that the acetone extract induced apoptosis in glioma cells. The extract induced reactive oxygen species generation and suppressed the expression of cell survival proteins. The migration of cancer cells was also suppressed by the acetone extract. The gas chromatography-mass spectrometry (GC-MS) analysis indicated that tetracontane, dotriacontane, hexatriacontane, pentacosane, hexacosane, and eicosane are the major components in the acetone extract. Collectively, the extract from C. raktakanda exhibited anti-carcinogenic activities in cancer cells. We are exploring whether the phytoconstituents, individually, or collectively contribute to the anti-cancer activities of C. raktakanda.Although over 100 species of Curcuma are reported, only  Glioblastoma multiforme  is the most aggressive, invasive, and most common tumor of the central nervous system . These tThe natural products have been used for the treatment of several illnesses including cancer since ancient times. Globally, 80% of the human population are estimated to rely on plant-derived medicines for their health care needs . FurtherCurcuma longa, the golden spice turmeric, is being extensively studied. Curcumin, a minor but the most active component of turmeric, is reported to exhibit activities both in animal models and in humans -2,5-diphenyl tetrazolium bromide (MTT) were obtained from SRL Diagnostics . The 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), 4′,6-diamidino-2-phenylindole (DAPI), 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carbocyanineiodide (JC-1), acridine orange, agarose, alexa fluor 488, ethidium bromide, fetal bovine serum (FBS), and propidium iodide were obtained from Invitrogen . Bcl-xL antibody was obtained from Santa Cruz Biotechnology  while GAPDH  was obtained from Abgenex . The human breast , cervical (HeLa), and rat glioma (C-6) cell lines were obtained from the National Centre for Cell Science (NCCS), Pune, India. MDA-MB-231, MCF-7, and HeLa cells were cultured in high glucose DMEM, while RPMI-1640 was used for C-6 cells. The FBS (10%), penicillin (100 units/mL), and streptomycin (100 µg/mL) were used to supplement the media. The mitochondrial reductase activity was measured to determine the effect of extracts on the viability of cancer cells using MTT as a substrate . The cytThe ability of a single cell to grow into a colony was examined by clonogenic assay, which is an in vitro cell survival assay. We used a method described previously with minor modifications . For thi+, Hercules, CA, USA).DNA laddering is a distinctive feature during the late stages of apoptosis. The assay was performed using a method described earlier . The celTo examine the effects of the extracts on the nuclear morphology, DAPI staining was performed . C-6 gliIn order to examine cell viability accurately, we performed AO/PI staining. C-6 cells were treated with the acetone extract (1–50 µg/mL). After 24 h, the cells were washed with PBS, stained with AO/PI, and visualized under the fluorescence microscope.We stained the cells with propidium iodide (PI) to examine the effects of the acetone extract on the sub-G1 population by flow cytometry . The C-6To examine the effects of acetone extract on the mitochondrial membrane potential, we used a fluorochrome JC-1 as reported earlier . BrieflyWhether the acetone extract affects the expression of cell survival proteins was examined by western blot analysis . The whoTo examine the effects of the acetone extract on cell migration, we used a wound healing assay. In brief, C-6 glioma cells were seeded into a culture dish and allowed to grow to 70% confluency. A wound was introduced into the monolayers using a sterile tip and washed to remove the cell debris. The cells were then allowed to grow in the culture media with or without the acetone extract. After 0, 12, and 24 h of treatment with acetone extract, the wound area was examined under the phase contrast microscope.2DCFDA) for 15 min in the dark. The stained cells were examined under fluorescence microscope and also by flow cytometry using FACScan. The analysis of flow cytometry data was carried out using Cell Quest software .Whether the acetone extract has the potential to generate reactive oxygen species (ROS) in the C-6 glioma cells was examined by flow cytometry . Cells wCurcuma raktaknda . The compounds were separated using nonpolar Rxi 5 Sil MS capillary column in full scan mode with injector mode-splitless and quadra pole mass selective detector (MSD). The injection temperature was set at 250 °C while GC-MS interface temperature at 250 °C. An aliquot of 1 μL volume was injected into the column. Helium was used as a carrier gas . Mass spectra were detected at 70 eV. The temperature programming was set as follows: column temperature was started from 60 °C (held for 2 min) and linearly increased by 5 °C/min to 200 °C (held for 2 min); after that it was increased by 3 °C/min to 220 °C (held for 1 min); further it was increased by 6 °C/min to 250 °C (held for 7 min). Total GC running time was 51.67 min. The compounds were identified by comparing mass spectra of each peak with NIST5 and WILEY libraries.Shimadzu Make GC-MS-TQ-8030 instrument  was used for the gas chromatography-mass spectrometry (GC-MS) analyses of different fractions of C. raktakanda that has demonstrated larvicidal activities. Most of the experiments were performed using C-6 glioma cell lines. We also used breast  and cervical (HeLa) cancer cell lines to determine the specificity of the extract. The underlying mechanism and the potential components from the extract were also examined. The overall goal of this study was to examine the anti-cancer potential of the extract from C. raktakanda. The cytotoxic potential of the extract was examined by measuring mitochondrial reductase activity using MTT as the substrate. We observed that the viability of C-6 glioma cells was suppressed by the three extracts in a concentration dependent manner  from the rhizomes of t manner A. Howevet manner B. Next, we examined the colony forming ability of C-6 glioma cells without and with AE treatment. Cells were exposed to 4, 8, 16, 32, and 64 µg/mL AE for 6 h. Cells were then washed and allowed to form colonies for 7 days. The extract significantly suppressed the number of colonies at a concentration as low as 16 µg/mL C. At 64 Apoptosis, a process of programmed cell death, is a normal physiological process. However, cancer cells have developed mechanisms to evade apoptosis. The induction of apoptosis selectively in cancer cells is one of the potential strategies of cancer therapy. Numerous assays were performed to examine the apoptosis inducing potential of AE. First, we examined the changes in the morphology of the cells by AO/PI dual staining. While AO can enter both live and dead cells, PI enters only dead cells with compromised mitochondria. Acridine orange stains all nucleated cells to generate green fluorescence. Propidium iodide stains dead nucleated cells to generate red fluorescence. Thus, the live nucleated cells produce green fluorescence, while dead nucleated cells produce red fluorescence. An increase in the concentration of AE was associated with a significant decrease in the number of viable cells. At a concentration as low as 10 µg/mL, features of early apoptosis such as membrane blebbing and nuclear condensation were observed after 24 h of treatment A. To conNext, we examined the sub-G1 population in the cells treated with or without AE. The treatment of C-6 cells with AE was associated with a modest increase in sub-G1 population A. One haThe glioma is characterized by local invasion. Before invasion, cells are detached from the primary tumor and migrate to the surrounding normal brain tissue. C-6 cells were exposed to different concentrations of AE  and the migration of the cells was examined in the normal and treated cells after 12 h and 24 h . In contCancer cells have the inherent property of increased ROS generation that can be exploited to induce ROS selectively. Next, we examined the potential of AE on ROS generation in C-6 glioma cells. The cells were exposed to different concentrations of AE for 1 h and stained with H2DCFDA. The ROS generation was measured by flow cytometry and also by fluorescence microscopy. The ROS generation in control and in 5 µg/mL AE treated cells was minimal . HoweverC. raktakanda in cancer cells other than glioma. For this, we used breast  and cervical (HeLa) cancer cell lines. Cells were exposed to different extracts of C. raktakanda and proliferation was examined by MTT assay. The three extracts suppressed the proliferation of cancer cells in a concentration dependent manner -phenanthrenone (8.33%). The ethyl acetate fraction contained mainly 2-phosphabicyclo[3.1.0]hex-3-ene, 6,6-dimethyl-2,3,4-triphenyl-, (endo)- (16%), desmethylnomifensine (12.6%), 1,1,6-trimethyl-3-methylene-2- cyclohexane (9.48%), and cobalt, hexamethylbenzene-pentamethylcyclopentadienyl (8.67%). The major components in the acetone fraction were reported to be tetracontane (38.53%), (-)--1-(2-hydroxyethyl)-2,5,5,8a-tetramethyldecahydro-2-naphthalenol (9.10%) and dotriacontane (6.16%). A previous study demonstrated the larvicidal activity of ktakanda . HoweverC. longa), very little is known about other Curcuma species. Curcumin, which constitutes 2–5% of turmeric, is one of the most active components [C. raktakanda exhibit anti-cancer activities is unknown. Therefore, the focus of the current study was to examine the biological activities of C. raktakanda against glioblastoma cells.Although much has been published on the golden spice, turmeric  extract further supports its negative effects on the survival of C-6 glioblastoma cells.We observed that the three extracts of f Bcl-xL . The Bclf Bcl-xL ,32. Addif Bcl-xL ,34. The The main cause of glioblastoma mortality is not the primary tumor but because the tumor invades locally. This leads to the ineffectiveness of current treatment options and recurrence of the tumor. The invasion involves detaching the cells from the primary tumor followed by migration in the surrounding normal brain tissue. In our observations, CR extract was found to suppress the migration of C-6 glioma cells. The process of migration and invasion involved the synthesis of proteases such as metalloproteinases that degrade extracellular-matrix (ECM) components selectively . WhetherMalus domestica containing pentacosane exhibited activities against C-6, A549 (lung carcinoma), CHOK1 (ovarian), and THP-1 (acute monocytic leukemia) cells [The extract used in the current study is a mixture of several components. The GC-MS analyses indicated that the three extracts are different in terms of phytochemical constituents. The major component of acetone fraction such as tetracontane, dotriacontane, and hexatriacontane has not been reported to exhibit anti-cancer activities previously. However, minor components of the acetone fraction such as pentacosane and eicosane are reported to exhibit anti-cancer activities. In a previous study, the essential oil from the leaves of a) cells . Similara) cells . Eucalypa) cells ,39,40,41We also observed that the CR extract inhibits the viability of not only glioblastoma cells but also breast cancer cells and cervical cancer cells. These observations suggest the broad-spectrum effects of the extract. We observed that AE induces ROS generation in C-6 glioblastoma cells. The role of ROS in the regulation of cancer associated cell signaling pathways is well established . FurtherA previous study demonstrated that the CR extract exhibit larvicidal activities . Thus, oThe development of drug resistance is a major hurdle in the therapy of several cancer types including glioblastoma . Our unpC. raktakanda exhibit anti-carcinogenic activities. The components from the extract responsible for all these activities remains to be elucidated. The generation of ROS by the extract may contribute to its anti-cancer activities. Our observations are important as very little is known about this plant. One limitation of the current study is that the actual component of the extract responsible for the observed anticancer effects is not known. However, with several components, the mixture can target multiple glioblastoma associated pathways. This may offer an advantage as the glioblastoma is caused by dysregulation of multiple genes and thus multi-targeting approach is required. We are currently working on the constituents and the underlying mechanism by which this plant exhibits broad-spectrum anti-cancer activities.In conclusion, our observations suggest that the rhizome extract from


Due to the localized nature of Charcot foot, systemically altered levels of inflammation markers can be difficult to measure. The aim of this study was to investigate whether it is possible to detect an arteriovenous (A-V) flux in any locally produced inflammatory biomarkers from an acute Charcot foot by comparing local and systemic measurements.  We included patients with acute diabetic Charcot foot. Blood was sampled from the vena saphena magna on the distal part of the crus bilaterally as well as from the arteria radialis. To minimize the A-V shunting effect, the feet were externally cooled with ice water prior to resampling. before: 7.25 versus 0.41 pg/mL, resp., p = 0.008; Δvalueafter: 10.04 versus 1.68 pg/mL, resp., p = 0.032). There were no differences in the fluxes for other markers of inflammation.  Both before and after cooling, the A-V flux of interleukin-6 (IL-6) between the Charcot feet and the arterial level was significantly higher than the flux between the healthy feet and the arterial level (Δvalue We have found an increased A-V flux of IL-6 in the acute diabetic Charcot foot compared to the healthy foot in the same patients. Charcot osteoarthropathy is a rare disorder manifesting with aseptic inflammation and hyperemia in and around load-bearing bones and tissues. The process is normally unilateral and leads to progressive, uncontrolled resorption and degeneration of bone mass, resulting in spontaneous fatigue bone fractures –4. WhileThe precise pathological mechanisms underlying Charcot foot are still not fully understood. However, it is dependent on relatively unimpaired lower limb blood flow and established peripheral neuropathy –11. It cRecent evidence suggests that the initial inflammation is provoked by repeated local microtrauma and dysregulated bone resorption –16, whicSeveral studies have reported changes in biomarkers of bone resorption and inflammation in individuals with Charcot foot –24, and α (TNF-α), interleukin-6 (IL-6), and interleukin-17 subtypes A, E, and F (IL-17A/E/F), as well as decreased levels of interleukin-1β (IL-1β) and interleukin-8 (IL-8).Related to this, studies have explored the possible relationship between interleukin levels and acute Charcot foot , 27–29 aκB (NF-κB) system, as described by Petrova and Shanahan [In addition, many individuals with Charcot foot also seem to have a degree of vascular calcification and inflammation , 31. ThiShanahan . A possiShanahan –35.However, the biomarkers in question might only be produced locally around the inflamed bones in the foot, which means that the signal on a systemic level can be difficult to register. Furthermore, a general systemic release of these biomarkers might happen in response to a number of inflammatory processes unrelated to the Charcot foot.Therefore, it is plausible that a stronger and more specific signal from an acute Charcot foot might be achieved by measuring the flux of a specific marker between the local venous concentration in the foot and the arterial concentration. Local sampling from the dorsal venous arch of the foot in acute Charcot feet has previously been done by Gough et al. and Pearson et al. , 23, altThe aim of this study was to investigate whether it is possible to detect a flux in any locally produced biomarkers from an acute Charcot foot by measuring the arteriovenous (A-V) difference.We included participants with acute Charcot foot, recruited at the Copenhagen Wound Healing Center at Bispebjerg Hospital, Denmark, and at the Steno Diabetes Center, Gentofte, Denmark. The participants were referred by specialists after thorough physical examination, full blood panel, X-ray, bone scintigraphy, and/or MRI. All participants were examined as close as possible to the reported outbreak of the acute Charcot symptom (<3 months).Exclusion criteria included no diabetes mellitus, temperature difference < 2°C between the feet, duration > 3 months, foot ulcers, prior foot surgery, new objective foot deformities, bilateral Charcot foot, infection in the foot, antiosteoporotic medication, arterial insufficiency, or foot or toe amputation on either side.To confirm that the Charcot foot still had a high activity on the day of examination as assessed by a locally elevated blood flow, this was measured in the feet with venous occlusion plethysmography . Foot teThe study was approved by the Regional Ethical Committee for Copenhagen.To measure the fluxes in biomarker production in the acute Charcot foot, blood was sampled from the vena saphena magna on the distal part of the crus above the ankle. This was done on both the affected (Charcot) side and the healthy side. Arterial blood was sampled from the a. radialis .The venous drainage of the foot happens primarily through the veins saphena magna and parva, while the deep veins only play a minor role. The saphena veins connect through the dorsal venous arch on the dorsal side of the foot. The dorsal venous arch collects blood from both superficial and deep veins in the foot, as well as from the networks rete venosus plantare and rete venosus dorsale pedis. The superficial and deep veins of the foot are linked by communicating perforant veins. The few valves present in these perforants are turned so that blood can only run from the deep to the superficial veins, thus helping with thermoregulation and pressure absorption. This means that parts of the drainage of the deep foot happen through the superficial veins, which can thus be sampled from a superficial vein on the lower leg –39.tice).A portion of the blood flow in the feet bypasses normal microcirculatory exchange by shunting directly through A-V anastomoses. This is in part a thermoregulatory effect and is thus more prevalent at higher skin temperatures . As the tstart) (tice).All three sites were sampled simultaneously (tstart) . Fluxes In the following, venous samples from the acute Charcot foot are denoted as CF(v) and venous samples from the non-Charcot foot are denoted as non-CF(v).The blood samples were centrifuged at 4°C and stored at −80°C. All samples were analyzed together at the end of the study.tstart samples were analyzed on a Bio-Plex System multiplex immunoassay screening panel . The panel used screened for IL-1β, IL-1RA, IL-6, IL-8, IL-17A, and TNF-α, with the best signals detected for IL-6 and IL-8.To estimate the existing interleukin profile and the highest relative concentrations, Β ligand (fsRANK-L), osteoprotegerin (OPG), IL-17F, sRAGE, and AGEs. The remaining samples were analyzed as part of the daily hospital sample routines. The accepted intraindividual sample CV for all assays was 20%. AGEs were measured with a Human sandwich ELISA AGE kit  .IL-6 was measured with an IL-6 Quantikine HS ELISA kit  .IL-8 was measured with a Human CXCL8 Quantikine kit  .IL-17F was measured with a Human IL-17F Platinum ELISA kit  .Assays for fsRANK-L, osteoprotegerin, and sRAGE were performed as previously described .All analyses were performed by Biolab, Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark. Special ELISA setups were used for IL-6, IL-8, free soluble receptor activator of nuclear factor kappa-α-level of <0.05 was considered significant. Normal distribution in data was tested using Shapiro-Wilks tests. No transformations were used. t-tests or paired t-tests were used for variance analysis between groups in normally distributed data sets. For data sets not normally distributed, nonparametric tests in the form of the Mann-Whitney rank-sum test were used, while Wilcoxon signed-rank tests were used for comparing paired samples.Data are presented as mean ± 1 SD or range unless otherwise noted. An Statistics and general data handling were done using IBM SPSS Statistics v. 23 by IBM Corporation, SIGMAPLOT v. 11.0.0.77 by Systat Software Inc., Microsoft Excel 2000 v. 9.0.2812 by Microsoft Corporation, and Apache OpenOffice 4.0.1 by the Apache Software Foundation.We included 5 patients with acute Charcot foot. In total, 22 patients were screened for inclusion. Of these, 7 patients were excluded due to having foot ulcers or receiving foot surgery and/or ulcer debridement before the examinations. Another 7 did not want to participate or were unable to participate due to personal reasons, while 3 patients had had their Charcot foot for too long to be considered acute (duration > 3 months).The average time from the reported onset of symptoms to examination was 7.2 weeks. The Charcot feet were on average 2.6°C warmer than the contralateral and had a 3 times increased blood flow. All 5 patients had recently started off-loading treatment with an AirCast® removable walker boot before measurements. All 5 patients were diagnosed with stage 0 Charcot foot.tstart). There were a significant higher temperature and blood flow in the acute Charcot feet compared to the healthy feet.Anthropometric data for the participants are listed in β, IL-1RA, IL-17A, and TNF-α, almost all measured values were below the multiplex limit of detection in all samples. This was tested with both serum and plasma. The detection limits were 1.32 pg/mL for IL-1, 29.64 pg/mL for IL-1RA, 7.99 pg/mL for IL-17A, and 12.72 pg/mL for TNF-α. There was a single signal in one patient in IL-1RA and IL-17A (not the same patient for both markers).For IL-1The average level of IL-6 detected was 10.6 pg/mL, and for IL-8, it was 12.5 pg/mL.tstart, there were no differences in the levels of fsRANK-L, OPG, IL-6, IL-8, sRAGE, or AGEs—neither between arterial and CF(v) nor between CF(v) and non-CF(v) samples. The highest relative numerical difference was for IL-6 in arterial versus CF(v) levels (7.31 versus 14.56) (p = 0.109). It was not possible to measure IL-17F as it was below the assay detection limit of 7.8 pg/mL for all samples at tstart and tice.Measurements from all three sites , and non-CF(v)) before and after cooling are listed in p = 0.008). There were no differences in fsRANK-L, OPG, IL-8, sRAGE, or AGEs.The venous-arterial flux of IL-6 between the Charcot feet and the arterial level was significantly higher than the flux between the healthy feet and the arterial level  , and the non-Charcot feet were cooled at an average of 12.9°C (from 31.1 to 18.2°C). Temperatures in each foot before and after cooling are listed in tice, there was a significantly elevated level of IL-6  in the Charcot feet compared to the arterial value (p = 0.049) (p = 0.002) (At = 0.049) . There w= 0.002) . There wtice was still significantly increased in the Charcot feet (CF(v)-arterial) compared to the healthy feet (non-CF(v)-arterial)  (p = 0.032). There were no differences in the fluxes for fsRANK-L, OPG, IL-8, sRAGE, or AGEs.The venous-arterial flux for IL-6 at tice was 3.7 in the arterial sample, 4.0 in the CF(v) sample, and 3.8 in the non-CF(v) sample and did not differ in a one-way ANOVA on ranks (p = 0.970).The fsRANK-L/OPG ratio at In this study, we have tested a novel approach to evaluating the local inflammatory activity in an acute Charcot foot by measuring the venous-arterial flux across the Charcot foot while lowering the possible dilution from A-V shunting by externally cooling the foot.The data show a difference in the venous-arterial flux of IL-6 both before and after external cooling. We also saw a two-fold elevated level of IL-6 in the Charcot foot compared to the arterial level after cooling, indicating a local production of IL-6. It is interesting that both IL-6 and AGEs only show a significant difference between the Charcot foot and arterial level after cooling, thus possibly indicating an effect of limiting A-V shunting in the feet before sampling.The results are mostly in line with what other groups have found. Divyateja et al. indicated an increased median IL-6 level in the Charcot foot , while bUnlike Folestad et al., we have been unable to demonstrate high levels of IL-17F systemically in acute Charcot patients . HoweverA finding of locally increased levels of IL-6 is of particular interest for several reasons. As a proinflammatory cytokine, its presence supports the theory regarding the pathogenesis of acute Charcot foot as put forth by, for instance, Jeffcoate et al. . Furtherα through RANK-L [Recently, Petrova et al. reported that OPG was elevated in patients with Charcot foot without a corresponding elevation in RANK-L  and thath RANK-L . It is ih RANK-L .Ndip et al. have indicated that individuals with Charcot foot have an increased RANK-L/OPG ratio and suggest that this could play a role in medial vascular calcification . We haveRegarding the increased level of AGEs after cooling, it is unclear whether this is an expression of a local increase in production of AGEs due to cooling, a by-product of the Charcot inflammation, or merely a random sampling variation.κB system and several associated cytokines [The presence of tissue-bound receptors for AGEs (RAGE) has been associated with impaired bone matrix mineralization and enhanced osteoclast formation . AGEs haytokines , 54. As To our knowledge, this is the first time that the local venous-arterial flux across an acute Charcot foot has been studied. Furthermore, we are unaware of other studies that have limited the local A-V shunting effect prior to measuring a Charcot foot.The study was limited by the number of available participants. In total, we screened 22 patients and most of these were excluded due to foot ulcers or extended time from the symptom onset to diagnosis. Thus, part of the recruitment issue was the rigorous exclusion criteria needed to ensure that any possible findings were not clouded by infections, surgery, or prolonged Charcot inflammation.Furthermore, most of the assays we have used have a limited accuracy and substantial intraindividual variations, and thus it was difficult to register any possible differences. These variations might be the reason why we saw an increase in AGEs after cooling in the Charcot foot compared to the arterial level. Unless more accurate assays are developed, future tests in a similar setup could be performed with multiple samples from each site and each time point to help alleviate this issue.In conclusion, we have found an increased venous-arterial flux of IL-6 in the acute diabetic Charcot foot compared to the healthy foot. We also found an increased level of IL-6 and AGEs in the acute Charcot foot compared to the arterial level after, but not before, externally cooling the feet.